Identification of ciliate grazers of autotrophic bacteria in ammonia-oxidizing activated sludge by RNA stable isotope probing.

It is well understood that protozoa play a major role in controlling bacterial biomass and regulating nutrient cycling in the environment. Little is known, however, about the movement of carbon from specific reduced substrates, through functional groups of bacteria, to particular clades of protozoa. In this study we first identified the active protozoan phylotypes present in activated sludge, via the construction of an rRNA-derived eukaryote clone library. Most of the sequences identified belonged to ciliates of the subclass Peritrichia and amoebae, confirming the dominance of surface-associated protozoa in the activated sludge environment. We then demonstrated that (13)C-labeled protozoan RNA can be retrieved from activated sludge amended with (13)C-labeled protozoa or (13)C-labeled Escherichia coli cells by using an RNA stable isotope probing (RNA-SIP) approach. Finally, we used RNA-SIP to track carbon from bicarbonate and acetate into protozoa under ammonia-oxidizing and denitrifying conditions, respectively. RNA-SIP analysis revealed that the peritrich ciliate Epistylis galea dominated the acquisition of carbon from bacteria with access to CO(2) under ammonia-oxidizing conditions, while there was no evidence of specific grazing on acetate consumers under denitrifying conditions.